Pharmaceutical and diagnostic compositions containing nanoparticles useful for treating targeted tissues and cells

ABSTRACT

Nanoparticles made from a select group of lipids and optionally containing a therapeutically active agent are employed in pharmaceutical compositions for delivery to targeted tissues and/or cells for the treatment or diagnosis of such diseases as cancer.

FIELD OF THE INVENTION

The present invention is generally directed to pharmaceuticalcompositions useful for targeting to tissues and cells for therapeuticand diagnostic purposes and methods for their preparation. Moreparticularly to non-gas-containing nanometer sized particles havingimproved cancer cell-targeting capacity and compositions containing thesame, optionally with at least one therapeutically active ordiagnostically useful agent.

BACKGROUND OF THE INVENTION

An ability to deliver therapeutically active agents to diseased tissuesand cells while avoiding damage to healthy tissues and cells, or theidentification of drugs that are pharmacologically selective for onetissue or cell type over another has presented a difficult andlong-standing problem for physicians treating patients. This isespecially true for cancer.

Cancer may be considered the result of rapid and endless division ofdiseased cells and the growth of cell clusters to form tumors. Malignantcells, spreading from a primary tumor mass and lodging elsewhere in thebody to form a secondary tumor burden. Differences between cancer cellsand healthy cells are subtle and historically most anticancerchemotherapeutic agents have sought to destroy tumor cells based on therapid and extensive cell division rate characteristic or cancer.

Examples of cell division related targets are DNA intercalation orcutting agents, replication, transcription and expression and repair orpolymerase enzyme activity inhibitors and microspindle polymerizationpoisons. Such agents include, but are not limited to, alkylating agents,antibiotics, antimetabolites, DNA intercalating agents, topoisomeraseinhibitors, taxanes, vinca alkaloids, cytotoxins, hormones,podophyllotoxin derivatives, hydrazine derivatives, triazinederivatives, radioactive substances, retinoids and nucleoside analogs(specific therapeutic agents include, for example, paclitaxel,camptothecin, doxorubicin, vincristine, vinblastine, bleomycin, nitrogenmustards, cisplatinum, 5-fluorouracil and their analogues). However,healthy tissues such as bone marrow and the epithelial lining of the gutfor example, also have rapidly dividing cell populations andchemotherapy agents typically fail to distinguish between these andother healthy and diseased cells. The result is dose limiting and evenlife threatening side effects that have become characteristic of cancerchemotherapy. For poorly aqueous soluble agents such as paclitaxel theuse of emulsifying agents such as Cremaphor has been suggested.Cremaphor has been shown to further contribute to the adverse sideeffect profile of paclitaxel.

One approach to making chemotherapy more selective for cancer cells isthe development of drugs that are based upon more recently discoveredbiochemical and metabolic differences between cancer and healthy cells.Such differences for example have now been described in receptor andsignal transduction pathways and oncogenes and gene regulators thatcontrol growth and differentiation or regulate apoptosis. Other examplesare tumor cell metabolic requirements for specific amino acids. Acutelymphoblastic leukemia cells for example are dependent on externalsources of the amino acid asparagine. The enzyme asparaginase has beenutilized to deplete circulating levels of asparagine in an attempt totreat disease. Newer classes of drugs, such as tyrosine kinaseinhibitors are being explored and with promising results. Tyrosinekinase activity has been linked to receptors such as epidermal growthfactor which may be upregulated in certain tumor types. Troublesome sideeffects and dose limiting toxicities as well as emerging drug resistancehave persisted and remained problems even with these more selectiveagents.

Additional differences between cancer and healthy cells have also beenobserved in the expression of cell surface antigens. Monoclonalantibodies and their fragments have been extensively studied for theselective diagnosis and therapy of cancer either by direct binding of anantibody to its antigen or the delivery of radioisotopes orchemotherapeutic agents that have been conjugated to the antibodybackbone. Typically monoclonal antibodies are specific for a limitednumber of cancer types and a “pancarcinoma” antibody has not yet beenidentified. Traditional cell division directed chemotherapeutic agentsas well as newer signal transduction directed agents and monoclonalantibodies or their fragments may fail to penetrate fully into a tumormass or accumulate sufficiently in tumor cells to achieve optimalresults (i.e. the active agent is not sufficiently internalized in thetumor cells). Such failures are usually associated with the physical orchemical features of the agents including charge, size, solubility,hydrophilicity, hydrophobicity, and other factors. Cure rates remainrelatively low for many solid tumor types and even modest improvementsin life expectancy are considered significant.

It has been reported that accumulation of a chemotherapy agent into atumor mass can be promoted by increases in the molecular mass of thechemotherapy agent. Lack of lymphatic drainage and other features oftumor associated vasculature such as leakiness are believed to play arole in this phenomena. Increases in molecular mass can be achieved bylipid acylation, conjugation to inert polymers such as polyethyleneglycol, polyglutamic acid, dextran and the like or by encapsulation ofdrugs into liposomes or nanoparticles of various sizes and compositions.Particles below 1 micron in size are believed to pass through the leakytumor vasculature and accumulate in the extracellular space of a tumormass. Polymer conjugation or encapsulation can also be utilized toimprove aqueous solubility or decrease plasma protein binding andaccumulation into healthy tissue.

Liposome encapsulated drugs such as doxorubicin are currently inclinical use for treatment of AIDS related Kaposi's sarcoma and ovariancancer. Polyethylene glycol or polyglutamic acid conjugated paclitaxeland polyethylene glycol conjugated camptothecin are presently in humanclinical trials.

In general, liposome or nanoparticle encapsulation and polymerconjugation while enhancing drug accumulation in a tumor mass mayactually slow or inhibit uptake or internalization of drug into tumorcells. Drugs are then left to diffuse out of degradable liposomes ornanoparticles. For polymer conjugation a prodrug strategy has beenadopted. Decreased rates of plasma clearance of these formulations hasalso been reported and suggested to contribute to increase tumor massaccumulation. In a prodrug strategy active drug is released from acarrier as the conjugate circulates through the blood.

One effort at addressing the issue of selective tumor destruction isdisclosed in U.S. Pat. No. 5,215,680. A moderately hydrophobic neutralamino acid polymer is labeled with a paramagnetic complex comprising ametal ion and organic chelating ligand. The labeled reagent is combinedin solution with a surfactant mixture and then shaken in a gaseousatmosphere to form a gas in liquid emulsion or microbubble. Althoughprincipally employed for the enhancement of ultrasonic and MRI imaging,mention is made of pooling or concentrating the microbubbles in tumorsto act as heat sinks as well as cavitation nuclei to disrupt the tumorstructure.

Another approach has been to develop solid lipid nanoparticles as adelivery system for drugs, including for sustained release or oraldelivery fromuations (See for example, Wolfgang Mehnert et al., “SolidLipid Nanoparticles, Production Characterization and Applications”, Adv.Drug. Del. Reviews, Vol. 47, pp. 165-196 (2001) incorporated herein byreference. However, delivery systems employing solid lipid nanoparticlesfor tumor targeting have been problematical at least in part because oflow drug-loading capacities, as well as unwanted accumulation in theliver and spleen or leaching of toxic agents remaining after particleformation.

Administering therapeutically active agents with an appropriate deliveryvehicle that would limit accumulation in healthy tissues while promotingaccumulation in a tumor mass and cellular internalization is highlydesirable. With more efficient delivery, systemic and healthy tissueconcentrations of cell division linked cytotoxic agents may be reducedwhile achieving the same or better therapeutic results with fewer ordiminished side effects. Such delivery of agents with inherent degreesof tumor cell selectivity would offer additional advantages. Further, adelivery vehicle that would not be limited to a single tumor type butwould allow for selective accumulation into a tumor mass and promotionof celluar internalization into diverse cancer cell types would beespecially desirable and allow for safer more effective treatment ofcancer. A delivery vehicle that would also allow for elevated loadingcapacity for the therapeutic agent would likely be a significant advancein the art.

Accordingly, there is a need for delivery vehicles which improve theefficiency of delivery of therapeutically active agents to targetedtissues including tumors with promotion of internalization into thetargeted cells (e.g. cancer cells) preferably without the need forchemical modification or conjugation of drug and which have hightherapeutic agent loading capacities. There is a further need for amethod of preparing and using such vehicles for delivery of a widevariety of therapeutically active agents to targeted tissues and cells.

SUMMARY OF THE INVENTION

The present invention is generally directed to delivery systems usefulfor the delivery of a particle having a desirable structure and particlesize distribution which may contain a therapeutically active agent totargeted tissues and cells of a warm-blooded animal including humans forthe prevention, diagnosis and/or treatment of diseases, conditions,syndromes and/or symptoms thereof, especially in the treatment ofcancer. In one aspect of the invention, a pharmaceutical composition isprepared incorporating non-gas containing particles optionallycontaining a desirable therapeutically active agent, especially for thetreatment and/or diagnosis of cancer in which the tumor cellsinternalize the composition to an extent significantly improved overprior particle delivery systems.

In one particular aspect of the present invention, there is provided apharmaceutical composition comprising non-gas containing particles inthe nanometer size range as hereinafter described and referred tohereinafter as “nanoparticles” comprising a mixture of a select group oflipids and optionally one or more therapeutically active agents. Theconcentration of the therapeutically active agent should be sufficientwithin the nanoparticles to provide effective internalization of thetherapeutically active agents selectively within a targeted tissueand/or cell.

In a further aspect of the present invention, there is provided non-gascontaining nanoparticles produced by a process comprising:

-   -   a) combining a mixture of a select group of lipids and        optionally at least one therapeutically active agent in an        organic solvent to form a solution;    -   b) adding the solution to an aqueous medium to form an aqueous        suspension; and    -   c) pertubating the aqueous suspension to form nanoparticles        within said aqueous medium.

In a further aspect of the invention, there is provided a non-gascontaining nanoparticle delivery system for the selective delivery ofthe particles optionally including at least one therapeutic agent totargeted tissues and/or cells comprising non-gas containingnanoparticles which are effectively internalized within targeted tissuesand cells, especially cancerous tissues and cells. The particles maycontain at least one therapeutic agent in sufficient concentration toallow effective internalization and concentration of the nanoparticlescontaining said at least one therapeutically active agent selectivelywithin the targeted tissue and/or cell and a pharmaceutically acceptablecarrier.

In a still further aspect of the present invention, there is provided amethod of selectively delivering the nanoparticles with or without atherapeutic agent into a targeted tissues and/or cells comprisingadministering to said targeted tissues and/or cells an effective amountof the non-gas containing nanoparticles comprising a mixture of lipidswith or without said therapeutically active agent as described herein.

In a further aspect of the invention, there is provided a method oftreating selected tissues and cells including cancerous tumors inwarm-blooded animals including humans by administering to saidwarm-blooded animals the pharmaceutical composition of the presentinvention as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not intended to limit the scope of the application asencompassed by the entire specification and claims.

FIG. 1A is a graph showing an embodiment of the particle sizedistribution of nanoparticles in accordance with the present invention;

FIG. 1B is a micrograph of cultured C₆ glioma cells incubated with andshowing internalization of fluorescent labeled nanoparticies of thepresent invention;

FIGS. 2A-2D are graphs showing particle size distribution ofnanoparticles loaded with paclitaxel, camptothecin, carmustine andetoposide, respectively;

FIG. 3 is a graph showing cellular internalization of the nanoparticlesshown in FIGS. 2A-2D in C₆ glioma cells;

FIGS. 4A-4D are graphs showing the effect of water-solubility enhancingagents on the particle size distribution of nanoparticles without atherapeutically active agent of the present invention;

FIG. 5 is a graph showing the effect of water-solubility enhancingagents on internalization of nanoparticles without a therapeuticallyactive agent of the present invention;

FIGS. 6A-6D are graphs showing the particle size distribution ofnanoparticles containing a water-solubility enhancing agent andcamptothecin as compared to nanoparticles with neither and nanoparticlesonly with camptothecin; and

FIG. 7 is a graph showing internalization of nanoparticles containingcamptothecin and a detergent as compared with nanoparticles containingneither and nanoparticles only with camptothecin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally directed to delivery systems foreffectively delivering non-gas containing nanoparticles optionallyincluding a therapeutically active agent to targeted tissues and/orcells for the prevention, diagnosis and/or treatment of a disease,condition, syndrome and/or symptoms thereof.

The delivery system is principally comprised of non-gas containingnanoparticles structured to achieve elevated passive accumulation aswell as active internalization into tumor tissues and cells. Targetedtissues and cells, especially tissues and cells associated withcancerous tumors readily internalize the nanoparticles and such elevatedinternalization levels coupled with high loading capacity of theparticles for the optional therapeutic agent provides a potent treatmentfor targeted tissues cells, including those associated with cancer.

The term “therapeutically active agent” as used herein includes anysubstance including, but not limited, to drugs, hormones, vitamins, anddiagnostic agents such as dyes, radioisotopes (e.g. P³², Tc⁹⁹, F¹⁸, I¹³¹and the like) and the like that are useful in prevention, diagnosis andtreatment of diseases, conditions, syndromes, and symptoms thereof,including cancer. The term “nanosized particles or nanoparticles” meansnon-gas containing particles of the nanometer range (i.e. from less than1 micron up to several microns or more) which are free of gas andtherefore are distinguished from microbubbles of the type described inthe U.S. Pat. No. 5,215,680.

The therapeutic agents useful for incorporation into the nanoparticlesinclude all types of drugs and in a particular aspect of the presentinvention includes cancer treating agents such as, for example,paclitaxel, carmustine, etoposide and camptothecin.

The nanosized particles are prepared by first forming a mixture of aselect group of lipids which provides the particles with a structurethat facilitates high internalization levels when applied to targetedtissues and cells. The lipid mixture generally comprises:

-   -   a) at least one first member selected from the group consisting        of glycerol monoesters of saturated carboxylic acids containing        from about 10 to 18 carbon atoms and aliphatic alcohols        containing from about 10 to 18 carbon atoms;    -   b) at least one second member selected from the group consisting        of sterol aromatic acid esters;    -   c) at least one third member selected from the group consisting        of sterols, terpenes, bile acids and alkali metal salts of bile        acids;    -   d) at least one optional fourth member selected from the group        consisting of sterol esters of aliphatic acids containing from        about 1 to 18 carbon atoms; sterol esters of sugar acids; esters        of sugar acids and aliphatic alcohols containing from about 10        to 18 carbon atoms, esters of sugars and aliphatic acids        containing from about 10 to 18 carbon atoms; sugar acids,        saponins; and sapogenins; and    -   e) at least one optional fifth member selected from the group        consisting of glycerol, glycerol di- or triesters of aliphatic        acids containing from about 10 to 18 carbon atoms and aliphatic        alcohols containing from about 10 to 18 carbon atoms.

The five members making up the lipid mixture are preferably combined ina weight ratio of (a):(b):(c):(d):(e) of 1-5:0.25-3:0.25-3:0-3:0-3.

In a particularly preferred form of the invention, the lipid mixture isformed from glycerol monolaurate, cholesterol benzoate, cholesterol,cholesterol acetate and glycerol palmitate which is principally in theform of glycerol tripalmitate.

While the lipid mixture described above only requires the presence ofthe first second and third members, it is preferred to incorporate thefourth and/or fifth members because their presence may improve stabilityand/or uniform particle size.

The nanoparticles formed from the lipid mixture of the present inventionare internalized into targeted tissues and cells, including canceroustumors to an extent sufficient to have a desired effect such as stoppinggrowth, inducing differentiation or killing the cell. The desired effectmay also include a diagnostic effect such as placing a detectable markerwithin the tissue or cell. “Internalization” as used herein means thatthe nanoparticles engage in active entry into the cell.

Factors that enable the nanoparticles to be selectively internalized bytargeted tissues and cells include not only the composition of the lipidmixture and the structure of the resulting nanoparticles but also thesize and molecular weight of the particles as described hereinafter.

The lipid mixture as described above may optionally be combined with adesired concentration of a therapeutically active agent. Typicalconcentrations of the therapeutically active agent can range from about1% or less to 30% or more by weight. The lipid mixture and thetherapeutically active agent are typically dissolved in a suitablesolvent (e.g. an alkanol such as ethanol).

The lipid solution with or without the therapeutically active agent isthen combined with water to form nanoparticles having a particle sizerange typically, but not always, in the range of from about 0.01 to 1.0microns. This range is particularly suitable for the treatment ofcancer. Larger particles may be appropriated for other uses. The rangeprovided herein will in part be determined by the lipid mixtureemployed, the type and amount of the optional therapeutically activeagent added to the lipid mixture and the presence or absence of an watersolubility enhancing agent such as a detergent as discussed hereinafter.

The nanoparticles formed within the aqueous medium (i.e. the aqueoussuspension) may then, according to need be treated to remove impuritiessuch as lipid materials, excess therapeutically active agent, solventsand the like to create a purified aqueous suspension suitable for use asa pharmaceutical composition for delivery to a warm-blooded animalincluding humans in need of treatment. In a preferred form of theinvention, the crude aqueous suspension is dialyzed to remove theimpurities and the dialysate is retained for pharmaceutical use.

In accordance with one aspect of the present invention, thenanoparticles are produced with a desirable particle size distribution,preferably where a major portion of the particles have a particle sizerange of from about 0.01 to 1 micron, preferably 0.1 to 0.5 micron withvarying minor amounts of particles falling above or below the range withsome nanoparticles ranging up to about 200 microns.

Reference is made to FIG. 1A showing a typical particle sizedistribution of nanoparticles produced in accordance with the presentinvention in accordance with the methods described in Examples 1 and 2.As shown specifically in FIG. 1A, the vast majority of the nanoparticlesin this embodiment are in the range of 0.1 to 1.0 micron although minoramounts of such nanoparticles range up to about 30 microns. As shown inFIG. 1B, the nanoparticles having the particle size distribution shownin FIG. 1A appear to be internalized within C₆ glioma cells as evidencedby the appearance of the nanoparticles throughout the cell except in thecell nucleus and confirmed by planar rotations analysis of cells usingconfocal microscopy.

As previously indicated, the particle size distribution of thenanoparticles will in part depend on whether a therapeutically activeagent is present and the type of therapeutically active agent which isincorporated therein. Referring to FIGS. 2A-2D there is shown theparticle size distribution of nanoparticles in accordance with thepresent invention containing paclitaxel, camptothecin, carmustine andetoposide, respectively. In each case, the vast majority of thenanoparticles are in the range of 0.1 to about 1.0 micron with varyingminor amounts of nanoparticles up to and including 200 microns.

As previously indicated, the lipid mixture-therapeutically active agentsolution is combined with water and then pertubated to producenanoparticles within the aqueous medium. The aqueous suspension may betreated to produce a purified liquid medium containing the nanoparticleswhich may be used for administration to warm-blooded animals. In someinstances, it may be desirable to remove unduly large particles, so asto better control the particle size distribution within a desired range.Suitable filtration systems such as from Millipore Corporation ofWaltham, Massachusetts are available for this purpose. The selection ofa suitable filter system therefore is a factor in controlling theparticle size distribution within a desirable range for thenanoparticles.

In accordance with one aspect of the present invention, thenanoparticles possess an elevated capacity for receiving thetherapeutically active agent (i.e. loading capacity) which makes theparticles particularly well suited for the selective delivery to andeffective concentration within cancerous tissues and cells.

The nanoparticles produced as described, when purified such as bydialysis to remove non-particulated drug, may be characterized todetermine the extent to which the nanoparticles may be internalized intargeted cells such as for example C₆ glioma cells.

Referring to FIG. 3, nanoparticles produced in accordance with thepresent invention as set forth in Example 2 containing one of the fourdrugs, paclitaxel, carmustine, etoposide and camptothecin were preparedas described above with non-incorporated drug removed by dialysisagainst distilled water. The nanoparticles were then incubated with C₆glioma cells cultured in 96-well plates at a seeding density of 10⁴cells per well in accordance with Example 4. After an appropriateincubation time the nanoparticle samples were aspirated from each welland the extent of cellular uptake (i.e. internalization) was plotted.

As shown in FIG. 3, each of the samples including the sample with nodrug was internalized within the C₆ glioma cells and therefore presentin the targeted cells to disrupt and possibly kill the targeted cells.

When the nanoparticles are prepared with a therapeutically active agent,the amount of agent which may be loaded into the particles will be anadditional factor in achieving a desirable effect on the targeted tissueand/or cells.

Referring to Tables I-IV, the amount of each of paclitaxel, carmustine,etoposide and camptothecin added to the lipid mixtures and the amountretained (loading capacity) after preparation as described above wascalculated as a function of the weight of the lipid mixture inaccordance with the method described in Example 3. Those results comparefavorably with known delivery systems of the prior art. TABLE IPaclitaxel Loading in Lipid Nanoparticles Paclitaxel added (percentAmount paclitaxel in Paclitaxel loading lipid weight) nanoparticles(μg/mL) (percent lipid weight)  1%  ND^(§) ND  5% ND ND 10% 20.84 10.42%20% 37.35 18.67% 30% 53.31 26.65%^(§)Level of drug was below detection limits.

TABLE II Carmustine Loading in Lipid Nanoparticles Carmustine addedAmount carmustine in Carmustine loading (percent lipid weight)nanoparticles (μg/mL) (percent lipid weight)  1% 3.13 1.57%  5% 3.461.73% 10% 4.84 2.42% 20% 14.60 7.30% 30% 25.41 12.71%

TABLE III Etoposide Loading in Lipid Nanoparticles Etoposide addedAmount etoposide in Etoposide loading (percent lipid weight)nanoparticles (μg/mL) (percent lipid weight)  1% 1.08 0.54%  5% 2.291.14% 10% 2.68 1.34% 20% 3.79 1.89% 30% 4.61 2.30%

TABLE IV Camptothecin Loading in Lipid Nanoparticles Camptothecin addedAmount camptothecin in Camptothecin loading (percent lipid weight)nanoparticles (μg/mL) (percent lipid weight)  1% 0.05 0.02%  5% 3.181.59% 10% 9.51 4.75% 20% 21.05 10.53% 30% 32.20 16.10%

The cells prepared as described above and after the determination ofdrug loading capacity as set forth in Tables I-IV were measured todetermine cellular internalization of the nanoparticles relative tonanoparticles in which no drug was incorporated.

As shown in FIG. 3, as compared to the control in which no drug wasincorporated, the amount of nanoparticles internalized into C₆ gliomacells were similar to or actually exceeded (i.e. paclitaxel andcamptothecin) internalization of the non-drug incorporatingnanoparticles. Thus, nanoparticles of the present invention appear toprovide dramatically improved internalization when loaded with drug andtherefore provide an effective system for the delivery oftherapeutically active agents for the treatment of targeted tissues suchas tumor tissues and cells.

In a preferred form of the present invention, it may be desirable toincorporate a water solubility enhancing agent and/or solvent into theprocess of preparing the nanoparticles in order to assure that the lipidmixture dispersed in the aqueous medium does not undesirably tend toprecipitate, thus reducing yield and possibly, delivery effectiveness ofthe therapeutically active agent.

To prevent or minimize the formation of a precipitate when producingnanoparticles containing a therapeutically active agent, the watersolubility enhancing agent may be incorporated when the lipid mixture iscombined with the therapeutically active agent. As a result, lesstherapeutically active agent is lost due to precipitation. Furthermore,maximizing water solubility may enhance the loading capacity of thelipid mixture while maintaining the desirable tumor targeting capabilityof the resulting product in a patient's body.

The optional water solubility enhancing agent may be selected from, forexample, solvents such as ethanol, acetonitrile, dimethylsulfoxide,chloroform, tetrahydrofuran, ether, dimethylformanide, diethylether andcombinations thereof. Other water soluble enhancing agents includedetergents (non-ionic, anionic and cationic), polyoxyethylene sorbitanfatty acid esters or polysorbates such as polyethylene oxide sorbitanmono-oleate, phospholipids such as phosphotidylcholine,phosphotidylethanolamine and phosphosphotidylserine. Other suitablewater soluble enhancing agents include polyoxyethylene alcohols,polyoxyethylene fatty acid esters, polyethylene glycol, polyethyleneglycol conjugated hydrophobic moieties, polyethylene glycol conjugatedlipids, ceramides, dextrans, cholates, deoxycholates and the like andmixtures thereof.

The water solubility enhancing agent when present may affect theparticle size distribution of the nanoparticles typically, but notnecessarily, increasing the particle size distribution. Referring toFIGS. 4A-4D, nanoparticles were prepared in accordance with the presentinvention by the method described in Example 5 (absent thetherapeutically active agent). Nanoparticles shown in FIG. 4A wereprepared without a water solubility enhancing agent while nanoparticlesshown in FIGS. 4B-4D included specific water solubility enhancing agents(i.e. detergents; deoxycholate, sodium dodecyl sulfate and Tween 80,respectively).

As shown in FIG. 4A, the mean particle size of the nanoparticlesprepared in the absence of detergent was 0.29 microns. When detergentwas added the mean particle size of the nanoparticles increased to arange of 0.43-0.46 microns.

The presence of a water solubility enhancing agent such as a detergentdoes not adversely affect internalization of the nanoparticles. As shownin FIG. 5, the internalization of the nanoparticles within C₆ gliomacells with detergent in accordance with the method described in Example6 were the same or similar to nanoparticles prepared in the absence ofdetergent.

The loading capacity of nanoparticles produced in accordance with thepresent invention is not adversely affected by the presence of a watersolubility enhancing agent. As shown in Table V, nanoparticles wereprepared incorporating 30% by weight of camptothecin alone or in thepresence of a detergent (i.e. 0.1% deoxycholate and 0.1% Tween 80). Asshown in Table V, loading capacity was enhanced for the detergentcontaining nanoparticles as compared with nanoparticles containing nodetergent. TABLE V Camptothecin Loading in Lipid Nanoparticles in thePresence of Detergents Amount Camptothecin Camptothecin camptothecin Inloading added (percent Detergent added (% nanoparticles (percent lipidweight) w/v) (μg/mL) lipid weight) 30% None 21.34 10.67% 30% 0.1%deoxycholate 31.25 15.62% 30% 0.1% Tween 80 30.59 15.30%

The nanoparticles produced with both a water solubility enhancing agent(e.g. a detergent) have a particle size distribution that provides foreffective internalization within targeted tissues and cells.

Referring to FIGS. 6A-6D there is shown a particle size distribution ofnanoparticles produced in accordance with Example 5. As shownspecifically in FIGS. 6C and 6D, the majority of the nanoparticlescontaining both a therapeutically active agent (camptothecin) and adetergent (deoxycholate and Tween 80, respectively) are in the range of0.1 to 1.0 micron. Amounts of such nanoparticles outside the range of0.1 to 1.0 micron have particle sizes up to about 80 to 100 microns.These results are similar to the nanoparticles having the particle sizedistribution shown in FIGS. 6A (no detergent and no camptothecin) and 6B(no detergent).

The nanoparticles described with reference to FIGS. 6A-6D were incubatedwith C₆ glioma cells in accordance with the method described in Example6. Cellular internalization of the nanoparticles in the C₆ glioma cellswas effective for each type of nanoparticle including those containingcamptothecin and detergents.

Emulsion enhancing agents may also be employed in the present inventionto ensure the formation of a desirable emulsion and the suspension ofthe non-gas containing nanoparticles in the aqueous media. Such emulsionenhancing agents include, but are not limited to, tricaprin, trilaurin,trimyrstin, tripalmitin, tristearin, Sofistan 142 as well as hard fats,glycerol, monostearate, glycerolbehenate, glycerolpalmitostearate,cetylpalmitate, decanoic acid, behenic acid, and Acidin 12. Otheremulsifying enhancing agents include soybean lecithin, egg lecithin,Poloxymer compounds (188, 182, 407 and 908), Tyloxapol, Polysorbate 20,60 and 80, sodium glycolate, taurocholic acid, taurodeoxycholic acid,butanol, butyric acid, diochtyl sodium sulfonsuccinate,monooctylphosphoric acid and sodium dodecyl sulfate.

When an emulsion enhancing agent is employed, it is preferred to combinethe emulsion enhancing agent with the lipid mixture and the optionaltherapeutically active agent in the initial preparation of thenanoparticles. In particular, the lipid mixture is mixed thoroughly withthe optional water solubility enhancing agent such as ethanol and thenagitated such as by sonication or by heating. The therapeutically activeagent is then added to the emulsion which has been combined with theemulsion enhancing agent under additional agitation for a timesufficient to complete dissolution or salvation.

As previously indicated, the pharmaceutical composition of the presentinvention is obtained by removing impurities such as by dialysis. Theresulting liquid medium (e.g. dialysate) provides a favorable means foradministering the nanoparticles to a warm-blooded animal. Dialysis is apreferred method of removing any non-particulated lipid mixturecomponents, drug and/or solvents and to achieve any desired bufferexchange or concentration. Dialysis membrane nominal molecular weightcutoffs of 5,000-500,000 can be used with 10,000-300,000 beingpreferred.

Suitable liquid media include injectable solutions or emulsions or othersuch liquid media suitable for administration by other pharmaceuticallyacceptable routes of administration which may contain, for example,suitable non-toxic, or acceptable diluents or solvents, such asmannitol, 1,3-butanediol, water, buffer solution, low carbon alcohol andwater solution, Ringer's solution, dextrose or sucrose solution, anisotonic sodium chloride solution, solutions in saline which maycontain, for example, benzyl alcohol or other suitable preservatives,absorption promoters to enhance bioavailability, and/or othersolubilizing or dispersing agents.

The pharmaceutical compositions of the present invention are generallysuitable as vehicles for the incorporation of substantiallylipid-soluble therapeutically active agents.

Accordingly, improved treatments of cancer are contemplated, includingtreatment of primary tumors by the control of tumoral cellproliferation, angiogenesis, metastatic growth, or apoptosis, andtreatment of the development of micrometastasis after or concurrent withsurgical removal, radiological or other chemotherapeutic treatment of aprimary tumor.

The pharmaceutical composition of the present invention increases theselectivity and specificity of delivery of therapeutically active agentsto a tumor mass through passive accumulation into tumor vasculature andactive internalization into tumor cells.

The invention provides methods for treating a patient withtherapeutically active agents and for delivering therapeutically activeagents to a cell for the prevention, diagnosis, and/or treatment ofdiseases, conditions, syndromes and/or symptom thereof. Thetherapeutically active agents employed in the present invention may beuncharged, or charged, nonpolar or polar, natural or synthetic, and thelike. In preferred embodiments of the present invention, thetherapeutically active agents may be selected from suitably lipophilicpolypeptides, cytotoxins, oligonucleotides, cytotoxic antineoplasticagents, antimetabolites, hormones, radioactive molecules, and the like.

Oligonucleotides as referred to above include both antisenseoligonucleotides and sense oligonucleotides, (e.g. nucleic acidsconventionally known as vectors). Oligonucleotides may be “natural” or“modified” with regard to subunits or bonds between subunits. Inpreferred embodiments, the oligonucleotide is a oligonucleotide capableof delivering a therapeutic benefit.

Antineoplastic agents are well known and include, for example, thefollowing agents and their congeners or analogues; camptothecin and itsanalogues such as topotecan and irinotecan, altretamine,aminoglutethimide, azathioprine, cyclosporine, dacarbazine,dactinomycin, daunorubicin, docetaxel, doxorubicin, gemcitabine,etoposide, hydroxyurea, irinotecan, interferon, methylmelamines,mitotane, paclitaxel and analogues such as docetaxel, procarbazine HCl,teniposide, topotecan, vinblastine sulfate, vincristine sulfate, andvinorelbine.

Other antineoplastic agents further include antibiotics and theircongeners and analogues such as actinomycin, bleomycin sulfate,idarubicin, plicamycin, mitomycin C, pentostatin, and mitoxantrone;antimetabolites such as cytarabine, fludarabine, fluorouracil,floxuridine, cladribine, methotrexate, mercaptopurine, and thioguanine;alkylating agents such as busulfan, carboplatin, cisplatin, andthiotepa; nitrogen mustards such as melphalan, cyclophosphamide,ifosfamide, chlorambucil, and mechlorethamine; nitrosureas such ascarmustine, lomustine, and streptozocin; and toxins such as ricin.

As used herein, an “effective amount of therapeutically active agents”means the dosage or multiple dosages at which the desired therapeutic ordiagnostic effect is achieved. Generally, an effective amount of atherapeutically active agent may vary with the subject's age, condition,dietary status, weight and sex, as well as the extent of the conditionbeing treated, and the potency of the drug being used. The precisedosage can be determined by an artisan of ordinary skill in the art. Thedosage may be adjusted by the individual practitioner in the event ofany complication. Generally, the nanoparticles will be delivered in amanner sufficient to administer and effective amount to the patient. Thedosage amount may be administered in a single dose or in the form ofindividual divided doses, such as from 1 to 4 or more times per day.

Dosage may be adjusted appropriately to achieve a desired therapeuticeffect. It will be understood that the specific dose level and frequencyof dosage for any particular subject may be varied and will depend upona variety of factors including the activity of the specifictherapeutically active agent employed, the metabolic stability andlength of action of that agent, the species, age, body weight, generalhealth, dietary status, sex and diet of the subject, the mode and timeof administration, rate of excretion, drug combination, and severity ofthe particular condition. Generally, daily doses of activetherapeutically active agents can be determined by one of ordinary skillin the art without undue experimentation, in one or severaladministrations per day, to yield the desired results.

In the event that the response in a subject is in sufficient at acertain dose, even higher doses (or effective higher doses by adifferent, more localized delivery route) may be employed to the extentthat patient tolerance permits. Multiple doses per day are contemplatedto achieve appropriate systemic or targeted levels of therapeuticcompounds.

Preferred subjects for treatment include animals, most preferablymammalian species such as humans, and domestic animals such as dogs,cats and the like, subject to disease and other pathological conditions.

A variety of administration routes for the pharmaceutical composition ofthe present invention are available. The particular mode selected willdepend, of course, upon the particular therapeutically active agentselected, whether the administration is for prevention, diagnosis, ortreatment of disease, the severity of the medical disorder being treatedand dosage required for therapeutic efficacy. The methods of thisinvention may be practiced using any mode of administration that ismedically acceptable, and produces effective levels of the activecompounds without causing clinically unacceptable adverse effects. Suchmodes of administration include, but are not limited to, oral,inhalation, mucosal, rectal, topical nasal, transdermal, subcutaneous,intravenous, intramuscular, or infusion methodologies.

The pharmaceutical compositions of the present invention may routinelycontain salts, buffering agents, preservatives, compatible carriers, andoptionally other therapeutic ingredients. When used in medicine, thesalts should be pharmaceutically acceptable, but non-pharmaceuticallyacceptable salts may conveniently be used to prepare pharmaceuticallyacceptable salts thereof and are not excluded from the scope of theinvention. Such pharmacologically and pharmaceutically acceptable saltsinclude, but are not limited to, those prepared from the followingacids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic,acetic, palicylic, p-toluene sulfonic, tartaric, citric, methanesulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzenesulfonic. Also, pharmaceutically acceptable salts can be prepared asalkaline metal or alkaline earth salts, such as sodium, potassium orcalcium salts of the carboxylic acid group.

The foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. One skilled in the art willreadily recognize from such discussion, and from the accompanyingclaims, that various changes, modifications and variations can be madetherein without departing from the spirit and scope of the invention asdefined in the following claims.

EXAMPLES Example 1 Preparation Of Nanoparticles Without TherapeuticAgent

10 mg of a lipid mixture containing a lipid selected from each of themembers of lipids specified herein in an amount meeting the specifiedweight ratio requirements was suspended in 1 mL of absolute ethanol. Theresulting suspension was added to 50 mL of distilled water and processedthrough a 110 Y Microfluidics Microfluidizer (Microfluidics, Inc.,Newton, Mass.) set at 85 psi air pressure at 25° C. and four passes.

The nanoparticles were collected and an aliquot examined for particlesize distribution using a Horiba LA-910 Laser Scattering Particle SizeDistribution Analyzer.

Example 2 Preparation Of Nanoparticles With Therapeutic Agent

10 mg of a lipid mixture containing a lipid selected from each of themembers of lipids specified herein in an amount meeting the specifiedweight ratio requirements was suspended in 1 mL of absolute ethanol. Astock solution containing one of the four drugs paclitaxel, carmustine,camptothecin and etoposide and absolute ethanol was prepared andcombined with the lipid mixture. The resulting suspension was added to50 mL of distilled water and processed through a 110 Y MicrofluidicsMicrofluidizer (Microfluidics, Inc., Newton, Mass.) set at 85 psi airpressure at 25° C. and four passes.

The nanoparticles were collected and an aliquot examined for particlesize distribution using a Horiba LA-910 Laser Scattering Particle SizeDistribution Analyzer. 15 mL of each preparation was concentrated washedtwice with water and dissolved in ethanol.

Example 3 Loading Capacity Of Nanoparticles

Nanoparticles produced in accordance with Example 2 were measured fordrug loading capacity in the following manner.

Calibration curves were established based on UV absorbance orfluorescence emission for each drug blanked against ethanol dissolvednanoparticles. More specifically: Etoposide, paclitaxel and carmustinewere monitored by UV absorbance at 254 nm, 230 nm, and 237 nm,respectively, using a Spectronic Genesys 5 photospectrophotometer.Camptothecin levels were determined by fluorescence monitoring at 390excitation and 460 emission using a microplate fluorimeter (FLUOstarOptima, BM Lab Technologies, Durham, N.C.).

Nanoparticles were prepared containing drug and dissolved in ethanolafter removal of any non-particulated drug by desalting centrifugationand washing using Millipore Ultrafree 2BHK40 with 100,000 nominalmolecular weight cutoff membrane and Eppendorf 5810R using rotor A-4-62at setting 3100 RPM for 90′ per wash.

Example 4 Internalization Of Nanoparticles in C₆ Glioma Cells

Nanoparticles prepared in accordance with Example 2 were additionallyprovided with 0.01% w/w of lipid cholesteryl BoPy FL C₁₂ (MolecularProbes, Eugene, Oreg.).

Samples (5 mL) were dialyzed (30 minutes) to remove any non-incorporateddrug and dye against distilled water (1.2 L) with two changes. C₆ gliomacells cultured in 96-well plates at a seeding density of 10⁴ cells perwell were seeded one day prior to each experiment and grown in completemedium consisting of DMEM and 10% fetal bovine serum.

The samples were diluted in complete media to 50 ug/mL and added to theC₆ glioma cells. The cells were incubated with diluted samples at 37° C.for 30 minutes. After appropriate incubation time, nanoparticle sampleswere aspirated from each well. Wells were then washed once by additionof 100 μL or PBS followed by aspiration and replacement of the samevolume of PBS.

The fluorescence intensity of the cells was quantified using amicroplate fluorimeter (FLUOstar Optima, BMG Labtechnologies, Inc.,Durham, N.C.).

Example 5 Preparation Of Nanoparticles Containing Detergent

Nanoparticles were prepared in accordance with the procedure of Example2 except that 200 μg/mL of lipids with the concentration of camptothecinand detergents shown in FIG. 6 were combined in a total volume of 50 mLin distilled water. Samples were prepared by passing the mixtures fourtimes through the Microfluidizer (Model 110 Y, Microfluidics, Inc.,Newton, Mass.). Collected products were analyzed on a laser scatteringparticle distribution analyzer (Model LA-910, Horiba, Inc., Ann Arbor,Mich.).

Example 6 Nanoparticle Internalization In C₆ Glioma Cells

Nanoparticle samples were each prepared with 200 μg/mL lipids, with theappropriate concentrations of camptothecin and detergents, and 0.01%cholesteryl BODIP Y-FL C₁₂ (w/w of lipids), in a total volume of 50 mLdistilled water. Samples were prepared by passing the mixtures fourtimes through the Microfluidizer (Model 110 Y, Microfluidics, Inc.,Newton, Mass.). Collected products were analyzed on a laser scatteringparticle distribution analyzer (Model LA-910, Horiba, Inc., Ann Arbor,Mich.). Flurorescent lipid nanoparticle samples were added to the C₆glioma cells, cultured in 96-well plates, at a concentration of 50 μg/mLin complete medium and incubated at 37° C. for 30 minutes. Samples weresubsequently removed, and cell monolayers were washed once with PBS. 100μL of PBS were added to each well, and nanoparticle uptake wasquantified by measuring fluorescence intensity of each well on amicroplate fluorimeter (FLUOstar Optima, BMG Labtechnologies, Durham,N.C.). Each sample was read in six separate wells, and the results wereaverage. Averaged values for each sample containing detergent werenormalized to averaged values of samples containing no detergents. Celluptake of each sample containing detergent, as quantified byfluorescence intensity, was expressed relative to that of sample with nodetergent.

1-13. (canceled)
 14. A pharmaceutical composition comprising non-gascontaining nanoparticles comprising: a mixture of select lipids capableof being internalized within a targeted tissue or cell sufficient toachieve a desired effect in the targeted tissue or cell, and apharmaceutically acceptable carrier.
 15. The pharmaceutical compositionof claim 14 wherein the mixture of select lipids comprises: a) at leastone first member selected from the group consisting of glycerolmonoesters of saturated carboxylic acids containing from about 10 to 18carbon atoms and aliphatic alcohols containing from about 10 to 18carbon atoms; b) at least one second member selected from the groupconsisting of sterol aromatic acid esters; and c) at least one thirdmember selected from the group consisting of sterols, terpenes, bileacids and alkali metal salts of bile acids.
 16. The pharmaceuticalcomposition of claim 15 wherein the nanoparticles further comprising atleast one lipid selected from the group consisting of: a) at least onefourth member selected from the group consisting of sterol esters ofaliphatic acids containing from about 1 to 18 carbon atoms; sterolesters of sugar acids; esters of sugar acids and aliphatic alcoholscontaining from about 10 to 18 carbon atoms, esters of sugars andaliphatic acids containing from about 10 to 18 carbon atoms; sugaracids, saponins; and sapogenins; and b) at least one fifth memberselected from the group consisting of glycerol, glycerol di- ortriesters of aliphatic acids containing from about 10 to 18 carbon atomsand aliphatic alcohols containing from about 10 to 18 carbon atoms. 17.The pharmaceutical composition of claim 15 wherein the lipid mixture hasa weight ratio of (a):(b):(c):(d):(e) of 1-5:0.25-3:0.25-3:0-3:.0-3. 18.The pharmaceutical composition of claim 14 wherein a major portion ofnanoparticles are in the range of from about 0.01 to 10 microns.
 19. Thepharmaceutical composition of claim 18 wherein the major portion of thenanosized particles are in the range of from about 0.1 to 5 microns. 20.The pharmaceutical composition of claim 14 wherein the nanoparticlesfurther comprise at least one water solubility enhancing agent.
 21. Thepharmaceutical composition of claim 20 wherein the water solubilityenhancing agent is a detergent.
 22. The pharmaceutical composition ofclaim 14 further comprise at least one emulsion enhancing agent.
 23. Thepharmaceutical composition of claim 14 wherein the targeted tissue orcell is a cancerous tissue or cell.
 24. The pharmaceutical compositionof claim 14 further comprising at least one therapeutically activeagent.
 25. The pharmaceutical composition of claim 24 wherein, the atleast one therapeutically active agent is a cancer treatingtherapeutically active agent.
 26. The pharmaceutical composition ofclaim 25 wherein the cancer treating therapeutic agent is selected fromthe group consisting of paclitaxel, carmustine, etoposide andcamptothecin and their congeners and analogues and combinations thereof.27-35. (canceled)
 36. The pharmaceutical composition of claim 24 whereinthe at least one therapeutic agent is selected from the group consistingof drugs, hormones, vitamins and diagnostic agents.
 37. Thepharmaceutical composition of claim 14 comprising a liquid mixturecontaining glycerolmonolaurate, cholesterol benzoate, cholesterol,cholesterol acetate and glycerol palmitate.